Immunoblot Analysis of Protein Expression

We collected HEK293T and neuronal cell lysates by harvesting the cultures in 150 µl lysis buffer (2% SDS, 1% Triton X-100, and 10 mM EDTA in PBS) supplemented with a protease inhibitor cocktail (PIC). Samples were boiled at 100 °C for 5 min after addition of 50 µl of 4x Laemmli sample buffer (BioRad, 1610747) containing 2-Mercaptoethanol (BME). Thirty µl of the boiled lysates were resolved on 13% SDS-PAGE gels and subjected to immunoblot analysis as described^{76 (link)}. Blots were probed in primary antibody (anti-GFP, mAb (clones 7.1 and 13.1), Roche (11814460001), 1:500; anti-syt1, mAb 48 (asv 48), Developmental Studies Hybridoma Bank, 1:500; anti-β-actin, mAb 3700 (8H10D10), Cell Signaling Technology, 1:500), diluted in 2.5% milk in TBST, overnight at 4 °C. Blots were washed thrice and incubated with a secondary antibody (Goat anti-Mouse IgG-HRP, 1706516, Bio-Rad Laboratories, 1:10 K), also diluted in 2.5% milk in TBST, for 1 h, then washed three times for a total of 15 min with TBST. Immunoblots were imaged using Luminata Forte Western HRP substrate (EMD Millipore; ELLUF0100) and a ChemiDoc MP Imaging System (Bio-Rad Laboratories). Bands were analyzed by densitometry, and contrast was linearly adjusted for publication using Fiji.